Rapid elimination of biotherapeutic molecules via renal clearance contributes to limited clinical effectiveness or more frequent dosing for the patient. Renal clearance due to glomerular filtration is most associated with smaller biotherapeutics, as the rates of kidney filtration are greatly reduced for molecules with a molecular weight of greater 50,000 daltons (Kontermann, Curr Opin Biotechnol 22:868-76, 2011). Several approved biotherapeutic drugs contain active portions that on their own fall below the filtration limit and are thus cleared quickly. To overcome this limitation, a number of technologies have been introduced to effectively increase the size of the therapeutic molecule to reduce kidney filtration and resulting half-life.
PEGylation (PEG) of therapeutics is an effective way to increase the hydrodynamic radius of the protein and reduce glomerular filtration. One or several PEG chains can be coupled to the protein most commonly through conjugation to free thiol or amine groups on the protein surface. PEGylated versions of Adenosine deaminase, L-Asparaginase, Interferon alpha-2b, G-CSF, Human Growth Hormone, Erythropoietin, Uricase, and an anti-TNFalpha antibody fragment have all been approved for human therapy (Kontermann, Curr Opin Biotechnol 22:868-76, 2011). Limitations of PEGylation include production of heterogeneous products and difficulty in controlling the number of PEG molecules attached to certain proteins. PEGylation introduces additional conjugation as well as purification steps to the production of therapeutic proteins, resulting in decreased yields and increased costs of goods. PEGylation may also lead to renal tubular vacuolization in animals and patients as PEG chains are non-degradable in the kidneys (Gaberc-Porekar et al., Curr Opin Drug Discov Devel 11:242-250, 2008).
Coupling a therapeutic to an antibody Fc region to generate Fc-fusion proteins can be used to increase the serum half-life of therapeutic molecules. Immunoglobulins may exhibit long half-lives on the order of several weeks in humans due to their large size and recycling through FcRn (Kuo et al., J Clin Immunol 30:777-789, 2010). TNF receptor 2, LFA-3, CTLA-4, IL-1R, and TPO-mimetic peptide molecules are all approved therapies produced as Fc-fusions (Kontermann, Curr Opin Biotechnol 22:868-76, 2011). Fc-fusion proteins are not ideal for all therapeutic classes for several reasons. The homodimeric nature of the Fc region results in the production of a dimeric therapeutic protein, possibly leading to cellular activation due to receptor clustering. Fc-fusions must also be made in mammalian expression systems which may be more costly than prokaryotic systems.
In addition to Fc, albumin exhibits a long half-life in vivo due to FcRn recycling. At a concentration of approximately 40 g/L, Human Serum Albumin (HSA) is the most abundant protein found in the blood. FcRn recycling leads to a long half-life of approximately 19 days in humans. Additionally, biodistribution studies suggest that albumin may distribute within the body to areas important for targeting disease, such as inflamed joints or tumors (Wunder et al., J Immunol 170:4793-4801, 2003). Thus, the serum half-life of a number of proteins has been increased by producing them as either C-terminal or N-terminal fusions to HSA. Successful fusions include interferon alpha (Flisiak and Flisiak, Expert Opin Biol Ther 10:1509-1515, 2010), human growth hormone (Osborn et al., Eur J Pharmacol 456:149-158, 2002), tumor necrosis factor (Muller et al., Biochem Biophys Res Commun 396:793-799, 2010), coagulation factor IX (Metzner et al., Thromb Haemost 102: 634-644, 2009), coagulation factor VIIa (Schulte, Thromb Res 122 Suppl 4: S14-19, 2008), insulin (Duttaroy et al., Diabetes 54:251-258, 2005), urokinase (Breton et al., Eur J Biochem 231:563-569, 1995), hirudin (Sheffield et al., Blood Coagul Fibrinolysis 12:433-443, 2001), and bispecific antibody fragments (Muller et al, J Biol Chem 282:12650-12660, 2007). HSA fusion proteins may have long serum half-lives, however large scale production of these fusion proteins is limited predominantly to yeast expression systems. Additionally, the large size of HSA may lead to a loss in activity of the therapeutic due to steric hindrance.
Therapeutic proteins may also be produced as fusion proteins to peptides or proteins that bind to serum albumin in the blood stream to increase their half life. Such albumin binding peptides include cysteine-constrained peptides or antibody fragments to albumin. Expression of a Fab antibody fragment as a fusion to cysteine-constrained peptides significantly increased the serum half-life of the Fab (Dennis et al., J Biol Chem 277:35035-35043, 2002; US2004/0253247A1). Coupling cysteine-constrained peptide to an antibody fragment led to better peak tumor accumulation and more homogeneous tumor distribution compared to Fab and mAb molecules targeting the same antigen (Dennis et al., Cancer Res 67:254-261, 2007; US2005/0287153A1). Further, a number of antibody fragments that bind specifically to albumin have been coupled to therapeutic moieties to increase the half life of the therapeutic. A camelid VHH antibody fragment (Nanobodies®) that binds to HSA was fused to another Nanobody® that binds to TNF-alpha (Coppieters et al., Arthritis Rheum 54: 1856-1866, 2006) or anti-EGFR Nanobodies® (Tijink et al., Mol Cancer Ther 7:2288-2297, 2008). Anti-albumin domain antibodies (dAbs) have been generated that bind to albumin, and have been fused to, for example, interleukin-1 receptor (Holt et al., Protein Eng Des Sel 21:283-288, 2008) and interferon alpha 2b (Walker et al., Protein Eng Des Sel 23:271-278, 2010) to improve their half life.
A number of naturally occurring protein domains from bacteria are known to interact with albumin, presumably to help such bacteria distribute throughout the host organism. These are 3-helix bundle protein domains approximately 6 kDa in size which use one face of the 3-helix bundle to interact with serum albumin (Cramer et al., FEBS Lett 581:3178-3182, 2007; Lejon et al., Acta Crystallogr Sect F Struct Biol Cryst Commun 64:64-69, 2008; Johansson et al., FEBS Lett 374:257-261, 1995; Johansson et al., J Mol Biol 266:859-865, 1997; Johansson et al., J Biol Chem 277:8114-8120, 2002). One such albumin binding domain derived from streptococcal protein G (Jonsson et al., Protein Eng Des Sel 21:515-527, 2008), has been most widely used to extend the serum half-life of proteins. Fusion to this domain has been shown to increase the half-life of soluble complement receptor type 1 (Makrides et al., J Pharmacol Exp Ther 277:534-542, 1996), a bispecific antibody (Stork et al., Protein Eng Des Sel 20:569-576, 2007), CD4 (Nygren et al., Vaccines 91:363-368, 1991; U.S. Pat. No. 6,267,964B1), Pf155/RESA (Stahl et al., J Immunol Methods 124:43-52, 1989), G-CSF (Frejd, F. PEGS Europe, Oct. 5, 2010), and affibody molecules binding to a number of targets (Andersen et al., J Biol Chem 286:5234-5241, 2011) (Frejd, F. PEGS Europe, Oct. 5, 2010). However, antibody production against the domain has been reported in patients and thus the use of the molecule for therapeutic applications may be challenging (Goetsch et al., Clin Diagn Lab Immunol 10:125-132, 2003; Libon et al., Vaccine 17:406-414, 1999).
A number of protein domains or peptides that bind to albumin are capable of extending the serum half-life and producing a more beneficial biodistribution pattern of therapeutic proteins. In order to use these albumin binding domains in therapeutic applications, a number of biophysical requirements need to be fulfilled, such as high expression levels in a host, solubility and stability, and minimal immunogenicity. The albumin binding moiety should bind to serum albumin with an affinity that effectively balances serum half-life and biodistribution with activity of the therapeutic moiety when bound and not bound to albumin.